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Genome-wide DNA methylation maps of mouse embryonic fibroblasts and reprogramming cells modulated by cell cycle and Dnmt1 expression

ABSTRACT: Dramatic change in DNA methylation patterns and levels both globally and/or locus-specifically is associated with the process of cell linage specification and somatic reprogramming. We found the expression of DNA methyltransferase 1 (Dnmt1) is tightly regulated by the features of cell cycle, which we referred as cell cycle-directed Dnmt1 expression adjustment. To further investigate how DNA methylation is affected by this mechanism, we applied reduced representation bisulphate sequencing (RRBS) to map genome-wide DNA methylation on mouse embryonic fibroblasts (MEFs). To characterize how this mechanism affects somatic reprogramming, samples of MEFs induced by Yamanaka factors (Sox2, Klf4, Oct4, cMyc) was included. The cell cycle was accelerated by shRNA targeted to p53, and the expression of Dnmt1 was manipulated genetically. We find 5mC level remains constant in regardless of cell proliferation rate, a result from the adjusted expression of Dnmt1. However, repressed expression of Dnmt1 in fast-proliferating cells results in global DNA demethylation. The patterns of DNA methylation and demethylation affected by this mechanism is sensitive to CpG densities. Generally, the results demonstrate cell cycle-directed Dnmt1 expression adjustment as an mechanism for the insurance of the stability of genomic 5mC inheritance.

ORGANISM(S): Mus musculus

PROVIDER: GSE93058 | GEO | 2017/10/13

SECONDARY ACCESSION(S): PRJNA359751

REPOSITORIES: GEO

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Project description:Transmission of 5-methylcytosine (5mC) from one cell generation to the next plays a key role in regulating cellular identity in mammalian development and diseases. While recent work has shown that the activity of DNMT1, the protein responsible for the stable inheritance of 5mC from mother to daughter cells, is imprecise; it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method that combines enzymatic detection of modified cytosines with nucleobase conversion techniques to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides. We find that the fidelity of DNMT1-mediated maintenance methylation is directly related to the local density of DNA methylation, and for genomic regions that are lowly methylated, histone modifications can dramatically alter the maintenance methylation activity. Further, to gain deeper insights into the methylation and demethylation turnover dynamics, we extended Dyad-seq to quantify all combinations of 5mC and 5-hydroxymethylcytosine (5hmC) at individual CpG dyads to show that TET proteins preferentially hydroxymethylate only one of the two 5mC sites in a symmetrically methylated CpG dyad rather than sequentially convert both 5mC to 5hmC. To understand how cell state transitions impact DNMT1-mediated maintenance methylation, we scaled the method down and combined it with the measurement of mRNA to simultaneously quantify genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from the same cell (scDyad&T-seq). Applying scDyad&T-seq to mouse embryonic stem cells transitioning from serum to 2i conditions, we observe dramatic and heterogenous demethylation and the emergence of transcriptionally distinct subpopulations that are closely linked to the cell-to-cell variability in loss of DNMT1-mediated maintenance methylation activity, with regions of the genome that escape 5mC reprogramming retaining high levels of maintenance methylation fidelity. Overall, our results demonstrate that while distinct cell states can substantially impact the DNA methylation maintenance machinery, there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state.

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Genome-wide DNA methylation maps of mouse embryonic fibroblasts and reprogramming cells modulated by cell cycle and Dnmt1 expression

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